Entry and infection of HIV-1 is initiated by the binding of its envelope glycoprotein (Env) to a receptor (CD4) and a co-receptor (predominantly CCR5 or CXCR4), and fusing its membrane with the host cell membrane. Understanding all aspects of the virus entry process and discovering alternative ways to modulate it remain important objectives for HIV research. Most efforts have largely focused on understanding the interactions of Env with CD4 and the chemokine receptors. In contrast, much less is known about the factors influencing the association of CD4 with the coreceptors preceding or during virus entry. Previously, we demonstrated a specific interaction between CD4 and CCR5, in the absence of Env, in both cell lines and primary human cells, and we and others have observed and reported on the possible existence of oligomeric coreceptors. We propose to define the molecular determinants of CD4 and coreceptor interaction, and determine the significance of coreceptor oligomerization, for tri-molecular complex formation (Env-CD4-corecptor) and Env-mediated fusion. Further, there is a consensus that clustering of a certain, but not precisely determined, number of CD4 and coreceptor molecules is necessary for fusion pore formation, and it has been suggested that a possible mechanism for facilitating clustering is present. Such a mechanism may be particularly important in primary cells where the numbers of CD4 and/or coreceptor molecules is much lower in comparison to cell lines; and related to this process is the observation that Env can induce a variety of intracellular, signaling events. These cellular responses may contribute to a receptor clustering mechanism and could also modulate post-entry stages of HIV-1 replication. We and others have observed that certain tyrosine kinase inhibitors can potently interfere with X4 HIV-1 mediated fusion and we have observed specific and potent inhibition, at the level of entry, of R5 HIV-1 infection of macrophages by these reagents. Understanding the significance of the signaling processes at play during virus infection and how the CD4 and coreceptor molecules interact together and with Env during infection will enhance our understanding of the entry and pathogenesis of HIV-1, and perhaps facilitate the discovery of new ways for prevention and treatment. We propose to examine, in detail, certain specific signaling-related events, which occur during virus entry and how the CD4 and coreceptor molecules interact and perhaps facilitate those events. Our Specific Aims are: 1) To define the molecular determinants of human CD4 and human CXCR4 and CCR5 interaction; 2) To determine the significance of CXCR4 and CCR5 oligomerization for HIV-1 entry; and 3) To understand the mechanism of the inhibition of HIV-1 fusion and virus entry mediated by tyrosine kinase inhibitors. We will utilize a variety of mutagenic approaches in conjunction with the use and characterization of a battery of non-human primate CD4 and CCR5 receptors; employ our assays of tri-molecular complex formation and analysis; and our assays for measuring fusion and virus entry.